Throughout this specification, various publications are referenced by Arabic numerals within parentheses. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this specification in order to more fully describe the state of the art to which this invention pertains.
Naturally occurring carboxypeptidase B Peptidyl-L-lysine (-L-arginine) hydrolase EC 3.4.17.2! is a zinc-containing pancreatic exopeptidase which specifically removes C-terminal Arg, Lys or Orn from peptides (1,2).
Naturally occurring rat carboxypeptidase B is produced from a precursor protein, preprocarboxypeptidase B, containing a 108 amino acid long N-terminal fragment which includes the signal sequence (13 amino acids) and an activation peptide (95 amino acids). Preprocarboxypeptidase B is enzymatically inactive.
During transport of preprocarboxypeptidase B to the endoplasmatic reticulum, the signal peptide is cleaved off; the resulting enzymatically inactive procarboxypeptidase B precursor is secreted from the cell. The enzymatically active carboxypeptidase B is then formed by cleavage of the activation peptide by trypsin (7).
Mature rat carboxypeptidase B contains 307 amino acids (5) and has an apparent molecular weight of 35 kD. It contains seven cysteine residues, six of which are paired into S--S bonds.
Carboxypeptidase B is widely used for commercial and research purposes, such as in the production of insulin and other biologically active polypeptides, and in protein sequence analysis.
Commercially available carboxypeptidase B purified from porcine pancreas is very expensive and is not totally free of other proteases.
The partial amino acid sequence of porcine precursor procarboxypeptidase B and the complete amino acid sequence of bovine carboxypeptidase B have been published (3, 4 respectively). In addition, the complete nucleotide sequence of the rat gene and the human cDNA have been published (5, 6 respectively).
Yamamoto et al. (6) have reported the recombinant expression of enzymatically inactive human procarboxypeptidase B lacking the first 11 amino acids of the activation peptide. They also report the recombinant expression of an enzymatically inactive .beta.-galactosidase-procarboxypeptidase B fusion protein wherein the procarboxypeptidase is lacking the first 11 amino acids of the activation peptide.
European Publication No. 588118 A2 discloses a bone-related carboxypeptidase-like protein named OSF-5. It is speculated that OSF-5 acts as an adhesion molecule or a growth factor and that it can be used as an agent for treating bone metabolic diseases. However, no actual function or activity for OSF-5 has been disclosed and no production of either naturally-occurring or recombinant biologically active protein has been demonstrated.
The subject invention discloses the production of recombinant, highly purified, enzymatically active and non-expensive carboxypeptidase B. Production of enzymatically active carboxypeptidase B has not been previously reported and the disclosure here is novel.